BRD4通过HMGB1/TGF-β1/Smad通路参与调控肺泡上皮细胞间质转化  

BRD4 regulates alveolar epithelial-mesenchymal transition through HMGB1/TGF-β1/Smad path

作　　者：陈茹茹 韩璐 何海兰 郝小惠 刘和亮 郭灵丽 Chen Ruru;Han Lu;He Hailan;Hao Xiaohui;Liu Heliang;Guo Lingli(School of Public Health,North China University of Science and Technology,Tangshan 063210;Hebei Key Laboratory of Organ Fibrosis,Tangshan 063210;College of Medical Technology,Chengdu University of Traditional Chinese Medicine,Chengdu 610075)

机构地区：[1]华北理工大学公共卫生学院,唐山063210 [2]河北省器官纤维化重点实验室,唐山063210 [3]成都中医药大学医学技术学院,成都610075

出　　处：《安徽医科大学学报》2025年第2期247-254,共8页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81602814);河北省自然科学基金(编号:H2017209154);河北省高等学校科学技术研究项目(编号:BJ2017006)。

摘　　要：目的探究溴结构域蛋白4(BRD4)在转化生长因子-β1(TGF-β1)诱导的肺泡Ⅱ型上皮细胞间质转化中的作用机制。方法采用不同浓度(5、10 ng/ml)的TGF-β1刺激MLE-12细胞48 h建立上皮间质转化(EMT)细胞模型,并对细胞予以50 nmol/L BRD4抑制剂JQ-1,100μmol/L高迁移率族蛋白B1(HMGB1)抑制剂甘草甜素(GA)和3μg/ml的重组高迁移率族蛋白1(rHMGB1)预处理。实验分组为:对照组、TGF-β1组、JQ-1组、JQ-1+TGF-β1组、GA组、GA+TGF-β1组、JQ-1+TGF-β1+rHMGB1组。采用CCK-8法检测JQ-1对细胞活力的影响;采用Western blot检测CDH1、ZO-1、Vimentin、α-SMA、BRD4、HMGB1、TGF-β1及Smad2/3、p-Smad2/3的蛋白表达水平;通过划痕实验检测细胞迁移能力。结果与对照组比较,TGF-β1组Vimentin、α-SMA蛋白表达增加,CDH1和ZO-1蛋白表达降低,提示EMT模型构建成功。在该模型中,BRD4、HMGB1的表达明显增加。不同浓度的JQ-1均可抑制MLE-12的细胞活力,且呈浓度依赖性。JQ-1和GA均可缓解TGF-β1诱导的EMT的发生,并抑制由TGF-β1引起的HMGB1表达的增加和TGF-β1/Smad2/3通路的激活,而采用rHMGB1上调HMGB1的表达可以减少JQ-1对EMT和TGF-β1/Smad2/3通路的影响。此外,JQ-1和GA均可以有效降低TGF-β1诱导的细胞迁移;而rHMGB1可以缓解JQ-1对细胞迁移速率的抑制作用。结论BRD4可通过影响HMGB1/TGF-β1/Smad2/3信号通路调控肺泡Ⅱ型上皮细胞向间质转化过程,且BRD4可能成为抑制肺纤维化的一个潜在靶点。Objective To investigate the mechanisms of bromodomain-containing protein 4(BRD4)in TGF-β1-induced epithelial-mesenchymal transition in alveolar type II epithelial cells.Methods MLE-12 cells were stimulated with different concentrations(5 ng/ml,10 ng/ml)of TGF-β1 for 48 h to establish an EMT cell model.The cells were pretreated with 50 nmol/L BRD4 inhibitor JQ-1,100μmol/L high mobility group box 1 protein(HMGB1)inhibitor glycyrrhizin acid(GA),and 3μg/ml rHMGB1.The experimental groups were divided as follows:control group,TGF-β1 group,JQ-1 group,JQ-1+TGF-β1 group,GA group,GA+TGF-β1 group,and JQ-1+TGF-β1+rHMGB1 group.The effect of JQ-1 on cell viability was examined using cell counting kit-8(CCK-8).The protein expression levels of CDH1,ZO-1,Vimentin,α-SMA,BRD4,HMGB1,TGF-β1,Smad2/3 and p-Smad2/3 were detected by Western blot.The cell migration ability was detected by a scratch test.Results Compared with the control group,the levels of Vimentin andα-SMA in the TGF-β1 group increased,and the levels of CDH1 and ZO-1 protein decreased,suggesting that the EMT model was successfully established.In this model,the expression of BRD4 and HMGB1 significantly increased.Different concentrations of JQ-1 could inhibit the cell viability of MLE-12 in a concentration-dependent manner.Both JQ-1 and GA could effectively alleviate TGF-β1-induced EMT,and reduce the increase in HMGB1 expression and the activation of TGF-β1/Smad2/3 pathway caused by TGF-β1.Moreover,rHMGB1 treatment could reduce the effects of JQ-1 on EMT and the TGF-β1/Smad2/3 pathway.Additionally,both JQ-1 and glycyrrhizin could effectively decrease TGF-β1-induced cell migration,whereas rHMGB1 could alleviate the inhibitory effect of JQ-1 on the rate of cell migration.Conclusion BRD4 can regulate epithelial-mesenchymal transition in alveolar type II epithelial cells via HMGB1/TGF-β1/Smad2/3 signaling cascade,and BRD4 may be a potential target for inhibition of pulmonary fibrosis.

关 键 词：溴结构域蛋白4 高迁移率族蛋白1 TGF-β1/Smad2/3 上皮间质转化 纤维化 JQ-1 

分 类 号：R135.2[医药卫生—劳动卫生]